For chemical synthesis of nucleic acid such as DNA, RNA and the like, a solid phase synthesis process using a phosphoramidite method is widely employed. In the solid phase phosphoramidite method, nucleic acid is generally synthesized by the following steps.
First, nucleoside to be the 3′ terminal of the nucleic acid to be synthesized is ester bonded to a cleaving linker such as succinyl group and the like via 3′-OH group so that the nucleoside is previously carried on a support for solid phase synthesis (nucleoside linker). Then, the support for solid phase synthesis on which the nucleoside linker is carried is placed in a reaction column which is then set on an automatic nucleic acid synthesizer.
Thereafter, a synthesis reaction comprising the following steps is generally performed in the reaction column according to a synthesis program of the automatic nucleic acid synthesizer:
(1) a step of deprotection of 5′-OH group of the protected nucleoside with an acid such as trichloroacetic acid/dichloromethane solution and the like;
(2) a step of coupling nucleoside phosphoramidite (nucleic acid monomer) with the deprotected 5′-OH group in the presence of an activator (tetrazole etc.);
(3) a step of capping an unreacted 5′-OH group with acetic anhydride and the like; and
(4) a step of oxidizing phosphite with aqueous iodine and the like.
By repeating the synthesis cycle, an elongation reaction of oligonucleotide from the 3′ terminal to the 5′ terminal direction is promoted, and a nucleic acid having a desired sequence is synthesized.
Lastly, a cleaving linker is hydrolyzed with aqueous ammonia, methylamine solution and the like to cleave the synthesized nucleic acid from the support for solid phase synthesis (non-patent document 1).
When the above-mentioned synthesis is performed, as mentioned above, it is necessary to carry, in advance, nucleoside to be the 3′ terminal (starting material) on a support for solid phase synthesis via a cleaving linker. Moreover, the 3′ terminal varies depending on the sequence of nucleic acid desired to be synthesized. In the case of DNA oligonucleotide, 4 kinds of dA, dG, dC, dT are necessary, and in the case of RNA, 4 kinds of rA, rG, rC, rU are also necessary. For synthesis of modified oligonucleotide, a support for solid phase synthesis previously carrying a modified nucleoside is necessary, making the process complicated.
To solve the aforementioned problems, a support for solid phase synthesis carrying a universal linker has been developed as a linker to connect a solid phase support and a starting material, in the place of nucleoside⋅succinyl linker and the like generally used heretofore. As used herein, the “universal linker” means a linker applicable irrespective of the kind of the 3′ terminal nucleoside. In the following, “a support for solid phase synthesis carrying a universal linker” is sometimes described as “universal support”. Using the universal support, the process includes, irrespective of the kind of nucleoside or nucleotide for the 3′ terminal of nucleic acid desired to be synthesized, reacting nucleoside phosphoramidite to be the 3′ terminal in the same step as general automatic nucleic acid synthesis to start the synthesis and, after synthesizing the desired nucleic acid, cleaving the nucleic acid from the support for solid phase synthesis by a method similar to a general method. It is not necessary to prepare a support for solid phase synthesis carrying various nucleoside-linkers as mentioned above.
For example, there are proposed some universal supports, which can synthesize a nucleic acid having a hydroxy group at the 3′ terminal (patent documents 1-5 and non-patent documents 2 and 3). The structure of these universal supports has two adjacent carbon atoms, one carbon atom being bound with —OH group to be the starting point of nucleic acid synthesis, and the other carbon atom being bound with a group (e.g., —OH group, —NH2 group, —SH group) to be a nucleophilic group upon removal of the protecting group. When the nucleic acid is cleaved by aqueous ammonia and the like after the nucleic acid synthesis, the protecting group of these nucleophilic groups are also dissociated to attack the 3′ terminal phosphorus, and the phosphate group is cleaved from the 3′ terminal to form cyclic phosphate. All are used to synthesize nucleic acid having a hydroxy group at the 3′ terminal.
Such nucleic acid having a hydroxy group at the 3′ terminal is highly useful since it is widely demanded in the biochemical field, such as a nucleic acid drug and the like.
However, conventionally-known methods using a universal support do not show a sufficient synthesis efficiency of DNA or RNA.
In view of such situation, a universal linker capable of inhibiting generation of byproducts and capable of synthesizing DNA or RNA more efficiently at a high purity, as well as a support for solid phase synthesis of nucleic acid which carries the linker have been demanded.